P21WAF1/CIP1 messenger RNA expression in hepatitis B, C virus–infected human hepatocellular...

p21WAF1/CIP1 Messenger RNA Expression in HepatitisB, C Virus–Infected Human HepatocellularCarcinoma Tissues

Susumu Kobayashi, M.D., Ph.D.

Kazuyuki Matsushita, M.D.

Kenichi Saigo, M.D.

Tetsuro Urashima, M.D.

Takehide Asano, M.D.

Haruyuki Hayashi, M.D.

Takenori Ochiai, M.D.

Second Department of Surgery, Chiba UniversitySchool of Medicine, Chuoh-ku, Chiba, Japan.

Supported by a Grant-in-Aid for Scientific Re-search from the Ministry of Education, Science,Sports and Culture in Japan.

Address for reprints: Susumu Kobayashi, M.D.,Ph.D., Second Department of Surgery, Chiba Uni-versity School of Medicine, 1-8-1 Inohana, Chuoh-ku, Chiba 260-8670, Japan; Fax: 81-43-226-2113; E-mail: [email protected]

Received May 30, 2000; revision received Decem-ber 18, 2000; accepted January 19, 2001.

BACKGROUND. The primary objective of this study was to clarify the significance of

p21WAF1/CIP1(p21) gene expression in the tumorgenicity of hepatitis B virus (HBV)

and hepatitis C virus (HCV) infected human hepatocelluar carcinoma (HCC).

METHODS. The authors performed Northern blot hybridization to compare the p21

messenger (m) RNA expression levels among 16 HCC cases. They detected tissue

HBVx mRNA (Northern blot) and plus- and minus-strand HCV RNA (reverse

transcription–polymerase chain reaction) in liver tissues. They also measured

alanine transaminase (ALT) levels and indocyanine green retention rate at 15

minutes (ICG-R15).

RESULTS. The p21 transcripts of tumor (T) tissues could be identified with lower

intensity than nontumor (N) tissues in all 4 HBVx mRNA(1) cases, 8 of 10 HCV

RNA(1) cases, and 1 of 3 B(2), C(2) cases (1 case was positive for both viruses).

p21 mRNA expression levels of N tissues were significantly higher in HCV RNA(1)

cases than in HBVx mRNA(1) cases. p21 mRNA expression levels of N tissues were

significantly correlated with serum ALT levels.

CONCLUSIONS. In HCV hepatitis, p21 mRNA expression is up-regulated to control

cell cycle under regeneration stress. Once the liver develops HCC, the p21 mRNA

expression decreases to prominently low levels. The up-regulated p21 expression

may play a role as a guard to prevent hepatocytes from tumorgenicity in HCV

hepatitis. Cancer 2001;91:2096 –103. © 2001 American Cancer Society.

KEYWORDS: p21WAF1/CIP1, hepatitis B virus, hepatitis C virus, hepatocellular carci-noma, Northern blot hybridization, mRNA, alanine transaminase (ALT), ICG-R15, cellcycle.

In 1993, p21WAF1/CIP1(p21) was identified as a human gene encodingCdk2 interacting protein (Cip1), in which induction was associated

with wild-type p53 (WAF1).1,2 It is now recognized that p21 protein isa universal inhibitor of cylin/Cdk activity and a mediator of growtharrest caused by p53-dependent and p53-independent pathways in-cluding the antimitogenic hormone-transforming growth factor–b

(TGF-b), oxidative stress, irradiation, and terminal differentiation.3–7

p21 protein induces G1 arrest of the cell cycle in which action isessential for differentiation and apoptosis.7–9 Therefore, the cells thatlost the activity to express p21 cannot regulate those cell cycles toattain G1 arrest for apoptosis and may lead to unregulated cell pro-liferation as a step toward carcinogenesis.

The hepatitis virus is frequently identified in patients with hep-atocellular carcinoma (HCC). Hepatitis B virus surface antigen(HBsAg) or anti– hepatitis C virus antibody (HCVAb) can be detectedin the sera of greater than 90% of patients with HCC. Therefore,

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© 2001 American Cancer Society

hepatitis B virus (HBV) or HCV has been considered asthe major putative cause of HCC in Japan.10,11 Approx-imately 70% of patients with HCC test sero positive foranti-HCV testings, with another 20% testing positivefor serum HBsAg.

In the mechanism of HBV-related hepatocarcino-genesis, HBV as a DNA virus is regarded as a nonse-lective mutagenic insertional agent, and the identifi-cation of a viral regulatory gene HBVx suggests thedirect involvement of HBV in oncogenesis.12 It hasbeen demonstrated that the HBVx protein transacti-vates viral and cellular genes involved in the transfor-mation.13 The HBVx protein also forms a complex withp53 protein, and evidence now is accumulating toindicate that HBVx may contribute to hepatocarcino-genesis by blocking p53 function. HBVx can bind theCOOH terminus of p53 and inhibit its DNA sequencespecific binding, its transcriptional transactivation,and apoptosis.14,15 The impairment of p53 gene maybe evoked not by the gene mutations but by the mech-anism through which this HBVx functions in HCC. Thedetriment of p53 function leads to the dysfunction ofp21. Wang et al. reported that HBVx negatively regu-lates the transcription of p21 by blocking p53 functionin their in vitro studies.16

Although serum HCVAb can be detected morefrequently than HBsAg in patients with HCC, studieson the HCV-related mechanism of HCC developmentrarely have been reported. However, it was reportedrecently that HCV core protein induced hepatocellularcarcinoma in transgenic mice.17 Ray et al. reportedthat the core protein down-regulated p53 promoteractivity18 and also repressed p21 promoter activitythrough the pathway of the p53 gene and of functionsother than p53.19 These events may be the direct rolesof HCV in the induction of HCC.

These data all are the results of in vitro studies,and the suppressive effect of these hepatitis viruses forp21 gene expression could be induced by the forciblyhigh expression of HBVx and HCV core protein usingvectors. In vivo, there are several possible situations inthe human liver, such as hepatitis and regeneration,which may influence p21 gene expression. If the in-fluence of hepatitis viruses and other factors is verifiedin human HCC tissues, it will be great step towarddisclosure of the mechanism of HCC development byHBV and HCV. In this study, we investigated whetherHBV and HCV infection really affect the mRNA expres-sion of the p21 gene or whether other factors have moreinfluence in clinically resected human HCC tissues.

MATERIALS AND METHODSPatients and HCC SpecimensThe subjects studied were 16 HCC cases who hadundergone hepatectomy at the Second Department of

Surgery of Chiba University Hospital. Frozen liverspecimens were surgically obtained from these 16HCC cases. Informed consent was obtained from thepatients for the use of their sera and resected speci-mens for the investigation concerned with hepatitis B,C infection, and RNA expression of the p21 gene. Thestudy protocol conforms to the ethical guidelines ofthe 1975 Declaration of Helsinki as reflected in a prioriapproval by the institution’s human research commit-tee. Routine pathohistologic examinations revealedthat all tumors were HCC, and that the nontumortissues were chronic hepatitis or cirrhosis. Paired sam-ples were obtained from tumor (T) tissues and corre-sponding nontumor (N) tissues. All samples were fro-zen in liquid nitrogen immediately after resection andstored at 280 °C until RNAs were isolated.

Presurgical Serum Tests for HBV, HCV, AlanineTransaminase, and ICG-R15The sera were obtained on admission for the detectionof HBsAg and HCVAb. Serum HBsAg was detected byenzyme immonoassay kits (Dainabot, Tokyo, Japan).The serum anti-HCV testing was performed usingHCV-passive hemagglutination kit (Dinabot, Tokyo,Japan).20 Serum alanine transaminase (ALT) activitymeasurements were performed on Hitachi 7600 au-tomat by using Wako Chemical reagents kit (WakoPure Chemical, Ltd., Tokyo, Japan). The commonthreshold of 45 IU/L was used as the upper normalrange. Indocyanine green tests were performed as themeasurement of the retention rate at 15 minutes aftervenous injection (ICG-R15). Serum indocyanine greenvalues were measured using a photoabsorbance in-strument (UV 1200; Shimadzu).

Reverse Transcription–Polymerase Chain Reaction forHCV RNAHepatitis C virus RNA was detected by the reversetranscription–polymerase chain reaction method afterthe extraction of RNA,21,22 as reported previously.23,24

The detection of plus-strand (sense) HCV RNA wasperformed by setting an outer antisense primer forreverse transcription. The minus-strand (antisense)HCV RNA was identified by switching the outer anti-sense primer to the outer sense primer for reversetranscription, as described previously.23

Northern Blot Hybridization for HBVx GeneThe Northern blot hybridization was prepared with 10mg total RNA obtained from T and nontumor N tis-sues. The RNA was electrophoresed on 1.0% agarosegels prepared in MOPS buffer (0.02 M morpholinopro-panesulfonic acid, 5 mM sodium acetate, and 1 mMethylenediamine tetraaetic acid, pH 7.0) containing 2.2M formaldehyde.25 After the identification of 28S and

Hepatitis Virus and p21WAF1/CIP1 in Hepatocellular Carcinoma/Kobayashi et al. 2097

18S of RNA stained by ethidium bromide, the denaturedRNA was transferred to nylon membrane filters (Amer-sham International, Buckinghamshire, UK).

The HBVx gene probe was prepared as an innersequence of 396 base pairs by use of PCR cloningstrategy for HBV subtype adr.26 The probe sequenceused for HBVx was shown in our previous report.27

The filters were incubated with these phosphorous32P-labeled (random priming) probes for 15 hours in asolution containing 50% formamide, 5 3 SSC, 53 Denhardt’s solution, 0.2 % sodium dodecyl sulfate(SDS), and 100 mg/mL salmon sperm DNA after pre-hybridization. The filter was washed in 2 3 SSC con-taining 0.2% SDS at 60 °C and then exposed to X-rayfilm.

Northern Blot Hybridization for p21In this study, we used a Cip1 cDNA donated by Dr. S.J.Elledge for the probe of p21.1 Membranes were pre-hybridized and hybridized to a 32P-labeled probe(Cip1 cDNA) in a same condition above. The filtersused for p21 were washed and rehybridized with glyc-eraldehyde-2-phosphate dehydrogenase (GAPDH) asthe control.

Relative p21 mRNA Expression LevelsThe levels of p21 mRNA expression were measured bydensitometric scanning for T or N tissues. The relative

p21 expression level first was calculated as each tissuelevel divided by a control tissue level. We determinedthe control tissue as the nontumor liver tissue of Case14, in which mRNA expression was measured on everymembrane (Tp21 or Np21 5 tissue p21/control p21).The GAPDH mRNA expression also was calculatedafter the same formula (TGAPDH or NGAPDH 5 tissueGAPDH/control GAPDH). All relative expression levelswere further calculated as T (5 Tp21/TGAPDH) in tumorand N (5Np21/NGAPDH) in nontumor tissues, respec-tively. Thereafter, we compared these levels betweentumor and corresponding nontumor (T vs. N) casesand among N of HBVx mRNA(1), HCV RNA(1), andB(2), C(2) cases. We also investigated the relationbetween the p21 mRNA expression levels of N tissuesand serum tests such as ALT and ICG-R15 values.

Statistical analyses were conducted using paired ttests for difference between T and N within the groupsand one-way analysis of variance (ANOVA) for differ-ences among T and N of the three groups. Post hocanalyses for ANOVA were performed by Bonferronitest. The relations between p21 mRNA expression lev-els of N tissues and serum ALT levels and presurgicalICG-R15 values were examined using Spearman cor-relation coefficient. Statistical calculations were per-formed using a computer statistical package (StatViewversion 5.0; SAS Institute Inc., Cary, NC). A P value lessthan 0.05 was considered significant.

TABLE 1Clinical Features and Summary of HBV, HCV Markers of HCC Patients

Patientno. Age (yrs) Gender

Diagnosis(HCC with)

HBV HCV

SerumHBsAg

Liver tissueN.B.H. for

HBVxSerumAnti-HCV

Liver tissue RT-PCRfor HCV RNA

T N T N

1 66 F CH 1 1 1 2 2 2 2 22 29 F CH 1 1 2 2 2 2 2 2 HBVx mRNA(1)3 65 M CH 1 1 1 2 2 2 2 24 67 F CH 2 1 1 1 1 1 1 15 55 M CH 2 2 2 1 1 1 1 16 78 M CH 2 2 2 NA 1 1 1 17 57 F Cirrhosis 2 2 2 NA 1 1 1 18 53 M Cirrhosis 2 2 2 1 1 1 1 1 HCV RNA(1)9 64 M Cirrhosis 2 2 2 1 1 1 1 110 58 M Cirrhosis 2 2 2 NA 1 1 1 111 66 M Cirrhosis 2 2 2 NA 1 1 1 112 62 M Cirrhosis 2 2 2 NA 1 2 1 113 57 M Cirrhosis 2 2 2 NA 1 1 1 114 40 M Cirrhosis 2 2 2 2 2 2 2 215 41 M CH 2 2 2 2 2 2 2 2 B(2), C(2)16 74 M CH 2 2 2 2 2 2 2 2

HBV: hepatitis B virus; HCV: hepatitis C virus; HCC: hepatocellular carcinoma; HBsAg: hepatitis B virus surface antigen; N.B.H.: Northern blot hybridization; RT-PCR: reverse transcription–polymerase chain reaction;

T: tumor; N: nontumor; CH: chronic hepatitis; NA: not available.

2098 CANCER June 1, 2001 / Volume 91 / Number 11

RESULTSHepatitis B, C MarkersThe clinical data of the 16 patients with HCC areshown in Table 1. The pathologic diagnoses were de-termined as chronic hepatitis for eight cases and cir-rhosis for another eight cases. Serum testings revealedthat three cases were HBsAg positive, and another 13cases were determined to be HBsAg negative. Anti-

HCV antibody could be detected in 4 HBsAg negativepatients among 10 serum-available cases.

The Northern blot analysis to detect HBVx tran-scripts revealed that the transcripts in T tissues couldbe identified in all 3 HBsAg positive cases (Cases 1, 2,and 3) and 1 case (Case 4) of 13 HBsAg negative cases.As shown in Figure 1, HBVx mRNA was dominantlyexpressed in T tissues compared with N tissues. Themolecular weight levels of expressed HBVx mRNAwere inconsistent and ranged between 2.0 and 9.0kilobases.23–25 The signal intensity also differed, ap-parently, from case to case. Case 4 (lane 4 in Fig. 1)was serum HBsAg negative; however, its signal showedthe highest intensity in all cases.

Reverse transcription–polymerase chain reaction todetect plus- and minus-strand HCV RNA revealed thatplus-strand HCV RNA could be identified in both T andN tissues among 10 of 16 cases; however, minus-strandHCV RNA could not be detected in T tissue of one case(Case 12) among plus-strand HCV RNA(1) cases. Usingthese results, we classified the 16 cases into 3 groups:HBVx mRNA(1), HCV RNA(1), and B(2), C(2) groups.HBVx mRNA(1), HCV RNA(1), and B(2), C(2) groupsconsisted of 4, 10, and 3 cases, respectively. One case(Case 4) was both HBVx mRNA positive and HCV RNApositive and was assigned to both groups.

Correlation between p21 mRNA Expression and HepatitisB, C Virus InfectionFigure 2 shows the Northern blot hybridization for p21mRNA expression, including ethidium bromide stain-

FIGURE 1. Northern blot hybridization of HBVx gene for RNAs from the liver

tissues of Cases 1, 2, 3, 4, and 6. T and N denote tumor and nontumor,

respectively. RNAs are shown in the photograph below after ethidium bromide

staining.

FIGURE 2. Northern blot hybridization

of p21WAF1/CIP1 for tumor (T) and nontu-

mor (N) in Cases 1, 3, 4, 6, 10, 12, and

14. Cases 1, 3, and 4 were HBVx mRNA

positive (HBVx mRNA[1]); Cases 4, 6,

10, and 12 were HCV RNA positive (HCV

RNA[11]); Case 14 was a both-nega-

tive case (B[2], C[2]). A control hybrid-

ization with GAPDH cDNA probe is

shown below the ethidium bromide

staining of RNAs. HBV: hepatitis B virus;

HCV: hepatitis C virus; Et.Br.: ethidium

bromide; GAPDH: glyceraldehyde-2-

phosphate dehydrogenase.


ing and GAPDH mRNA expression in HCC T and Ntissues in Cases 1, 3, 4, 6, 10, 12, and 14. The N tissuesof Cases 6, 10, and 12, which were exclusively HCVRNA positive, indicated a high level of p21 mRNAexpression, compared with other cases and the corre-sponding T tissues. Figure 3 shows relative p21 mRNAexpression levels in T tissues and N tissues (T 5 Tp21/TGAPDH; N 5 Np21/NGAPDH). The cases were classifiedinto three groups: HBVx mRNA(1) group, HCVRNA(1) group, and both negative (B[2], C[2]) group.Blot data were obtained for T tissue and corre-sponding N tissue of those three groups. In all 4cases in the HBVx mRNA(1) group, 8 of 10 HCVRNA(1) cases, and 1 of 3 B(2), C(2) cases, the p21mRNA expression level of T tissue was found to belower than that of corresponding N tissue. Themean plus or minus standard deviation values ofp21 mRNA expression levels were as follows: T5 0.18 6 0.05, N5 0.45 6 0.13 in the HBVx mRNA(1)group; T 5 0.58 6 0.48, N 5 1.93 6 1.15 in the HCVRNA(1) group; T 5 1.57 6 0.76, N 5 0.97 6 0.15 in

the B(2), C(2) group. As shown in Figure 3, p21mRNA expression levels of T tissues were signifi-cantly lower than N tissues in the HBVx mRNA(1)and HCV RNA(1) groups (T vs. N; paired t test). Therelative p21 mRNA expression levels in N tissues (N5 Np21/NGAPDH) were significantly higher in HCVRNA(1) than HBVx mRNA(1) groups (ANOVA).

Correlation between p21 mRNA Expression andPresurgical Serum ALT Levels and ICG-R15 ValuesFigure 4 shows the relation between p21 mRNA ex-pression levels of N tissues and presurgical serum ALTLevels. p21 mRNA expression levels of N tissues weresignificantly correlated with serum ALT levels (Spear-man correlation coefficient, 0.552; P 5 0.033). Figure 5shows the relation between p21 mRNA expression lev-els of N tissues and presurgical ICG-R15 values; how-ever, there was no significance in correlation betweenthe p21 mRNA expression levels of N tissues and pre-surgical ICG-R15 values (Spearman correlation coeffi-cient, 0.376; P 5 0.146).

FIGURE 3. Relation among relative p21WAF1/CIP1 mRNA expression ratios (p21WAF1/CIP1/GAPDH) in tumor (T) and nontumor (N) tissues of the three groups (HBVx

mRNA [1]; HCV RNA [1]; and B[2], C[2]). Before this estimation of relative p21WAF1/CIP1 mRNA expression ratios, we calculated the tissue/control tissue ratio for

p21WAF1/CIP1 and GAPDH. The control tissue was N of Case 14, which is indicated with an arrow. The striped circles denote Case 4, which was both HBVx mRNA

positive and HCV RNA positive. Relative p21WAF1/CIP1 mRNA expression 5 Tp21/TGAPDH or Np21/NGAPDH (Tp21 or Np21 5 tissue p21WAF1/CIP1/control p21WAF1/CIP1; TGAPDH

or NGAPDH 5 tissue GAPDH/control GAPDH). T versus N in HBVx mRNA(1) and HCV RNA(1), P , 0.05; N of HBVx mRNA(1) versus N of HCV RNA(1), P , 0.05.

NS: not significant; GAPDH: glyceraldehyde-2-phosphate dehydrogenase; HBV: hepatitis B virus; HCV: hepatitis C virus.


DISCUSSIONp21 first was isolated as one of Cdks interacting pro-teins (CIP1), which is a potent and tight binding in-hibitor of Cdks.1 The discovery of p21 gene has pro-vided not only a lucid access to negative regulation ofthe cell cycle as an inhibitor of cyclin–Cdk complex,but also a direct link between tumor suppressor pro-tein and the cell cycle because a gene, named WAF1,that had an identical cDNA sequence was identifiedsimultaneously and was induced by wild-type p53 butnot mutant p53 gene expression.2 In addition to cellcycle regulation and tumor suppression mediated byp53, p21 is involved in many other functions includingdifferentiation and apoptosis.7–9 Recently, Albrecht etal. reported that p21 expression is regulated by p53-dependent and -independent pathways in the liverand is up-regulated in a biphasic manner with en-hanced mRNA expression during G1-phase and afterS-phase according to hepatic regeneration after partialhepatectomy.28 Crary and Albrecht and Kane et al.also reported in other articles that p21 expression isup-regulated in hepatitis condition of HCV-infectedliver.29,30

In this study, by using surgical HCC specimens,we demonstrated that p21 mRNA expression was sup-pressed in most T tissues infected with hepatitis B, C

viruses as compared with the corresponding N tissues.As a reason for the decreased p21 mRNA expression ofT tissues of human cancers, p53 gene mutations aremostly suspected. However, Greenblatt et al. reportedthe low frequency of p53 mutations in HCC, in whichHBVx protein is detected.14 The influence of HBVxprotein for p53 protein has been well documented.Recent studies on transgenic mouse tumor modelssuggest that HBVx protein may contribute to hepato-carcinogenesis by binding to and cytoplasmic seques-tration of the cellular growth suppressor proteinp53.31,32 HBVx can bind to the COOH terminus of p53and induce the conformation of p53 protein. Thisevent inhibits the DNA sequence specific binding,transcriptional transactivation, and apoptosis as awild-type p53 function.14,32 This impaired p53 func-tion by HBVx protein eventually may negatively con-trol the transcription of p21.16 In a current study,HBVx transcripts could be detected in four cases, in-cluding three HBsAg positive cases and one negativecase, in which tissues p21 mRNA expression level of Ttissues was found to be lower than that of correspond-ing N tissues. The continuously expressed HBVx, whichpossibly had been deduced from the integrated HBVxgene, might reduce p21 mRNA expression by blockingthe p53 function, as in the reported in vitro studies.16

FIGURE 4. Relation between relative

p21WAF1/CIP1 mRNA expression ratios

(p21WAF1/CIP1/GAPDH) in nontumor (N)

tissues and serum alanine aminotrans-

ferase (ALT) levels. The calculation for

the relative ratio of p21WAF1/CIP1 was

performed following the method in Fig-

ure 3. HBVx mRNA(1) (unfilled circles);

HCV RNA(1) (filled circles); B(2), C(2)

(filled squares). The striped circle de-

notes Case 4, which was both HBVx

mRNA positive and HCV RNA positive

(Spearman correlation coefficient, 0.552;

P 5 0.033). GAPDH: glyceraldehyde-2-



These observations suggest that continuous expressionof HBVx may be the primary initial requirement in mul-tistep carcinogenesis of HCC, and HBVx binding to p53may be the possible mechanism for p53 inactivationamong HBV positive cases.14 However, this event is oneof the causes that decrease p21 expression in T tissues,because the impairment of p21 expression in T tissuescould be detected in most HCV positive cases.

Among 8 of 10 HCV RNA-detected cases, the p21mRNA expression level of T tissues also was found tobe lower than that of corresponding N tissues. Ray etal. demonstrated by using a cell culture system thatHCV core protein down-regulated p53 promoter ac-tivity,18 and they also described how HCV core proteinrepressed p21 promoter activity through a pathwayother than the p53 function.19 However, HCV can rep-licate through minus-strand HCV RNA more effi-ciently in N than T tissues, in which HCV produces thecore protein to yield new virus particles.23,33 Therecould be either no difference or more production ofthe HCV core protein in N tissues compared with Ttissues. Hence, in vitro studies, which have shown thatHCV potentially repressed p21 expression, cannot ex-plain the relatively large amount of p21 transcripts inN tissues of HCV-infected HCC. We examined therelations between p21 mRNA expression levels of Ntissues and serum ALT levels and presurgical ICG-R15values. We selected ALT for an indicator of inflamma-

tion of liver and ICG-R15 for liver functional damageas the results of fibrosis. The ALT and ICG-R15 areboth at high levels among HCV-infected patients withHCC. Conversely, they were relatively low among HBVpositive cases. p21 mRNA expression levels of N tis-sues were significantly correlated with serum ALT lev-els; however, we could not find the significance be-tween p21 mRNA expression levels of N tissues andpresurgical ICG-R15 values. These observations sug-gest that HCV-induced hepatic injury may up-regulatep21 gene expression of liver tissues as shown in thereport by Albrecht et al., and this influence mightconquer the repressive effect of HCV as confirmed byin vitro studies.19 In contrast with HCV positive cases,liver inflammation as indicated by ALT levels couldnot be identified in HBV positive cases. This mildinflammation might be the reason for the moderateelevation of p21 expression in N tissues of HBV posi-tive patients.

Among HBV positive cases, HBVx-induced repres-sion of p53 and p21 genes may be the primary initialrequirement in multistep carcinogenesis of HCC,which does not accompany chronic inflammation andelevated p21 expression in N tissues. In HCV positivecases, the up-regulation of p21 expression may beindispensable for the control of the cell cycle to attainG1 arrest and apoptosis under the HCV hepatitis andregeneration stress. In hepatitis, highly expressed p21

FIGURE 5. Relation between relative

p21WAF1/CIP1 mRNA expression ratios

(p21WAF1/CIP1/GAPDH) in nontumor (N)

tissues and indocyanine green retention

rate at 15 minutes after venous injection

(ICG-R15). The calculation for the rela-

tive ratio of p21WAF1/CIP1 was performed

following the method in Figure 3. HBVx

mRNA(1) (unfilled circles); HCV RNA(1)

(filled circles); B(2), C(2) (filled

squares); The striped circle denotes

Case 4, which was both HBVx mRNA

positive and HCV RNA positive (Spear-

man correlation coefficient, 0.376; P

5 0.146). GAPDH: glyceraldehyde-2-



may hold hepatocytes against transformation by in-ducing enough G1 span to evoke apoptosis or repairDNA mismatches under the activated cell cycle pro-gression. For the long duration of chronic hepatitisespecially induced by HCV, the repeated death andregeneration of hepatocytes increase the chances ofDNA arrangement, which may make it impossible forhepatocytes to keep p21 expression on high level andeventually lead to the tumorgenicity as HCC. In HCVhepatitis patients, this up-regulation of p21 expressionmay play an important role as a guard to preventhepatocytes from malignant change for HCC.

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